Although recent studies demonstrate that humans can generate new heart cells, the rate of renewal dramatically decreases with age. Since heart failure often strikes in the second half of life, understanding how heart cells regenerate and why this process declines with age is a critical goal. The inducible cre-lox approach is a widely-used method for fate-mapping of cells in mammals. In this proposal, we show preliminary data revealing that an inducible cre-lox approach to genetically label cardiomyocytes in mice is not suitable for studying the aging process in the myocardium. Our preliminary data reveal age-related recombination in cardiomyocytes in the absence of induction of the cre activity. Thus, we have developed an entirely new approach to fate-mapping using isotopes in a cell-specific metabolic precursor. This metabolic fate-mapping approach utilizes an isotope-enriched metabolic tracer, specifically creatine, which is taken up by cells and utilized in the cytoplasmic phosphocreatine shuttle. The intracellular creatine pool is known to turn over at a rate of approximately 2-3% per day, making it an ideal cardiomyocyte-specific metabolic label. Cells that are creatine positive can be identified via use of Multi-Isotope Imaging Mass Spectrometry (MIMS), a high resolution quantitative approach. Using this new metabolic fate-mapping technique, the specific aims for this study are: 1. To test the hypothesis that, together with MIMS, metabolic fate-mapping using isotope-labeled creatine will enable the detection of cardiomyocyte regeneration during normal aging for mice of all age groups. We anticipate that the rate of regeneration will be low, and this basal cell division rate will decrease as a function of age. 2. To test the hypothesis that, together with MIMS, metabolic fate-mapping using isotope-labeled creatine will enable the detection of cardiomyocyte regeneration after injury for mice of all age groups. We anticipate that regeneration will primarily be from stem cell differentiation and not by pre-existing cardiomyocyte cell division, and this rate of regeneration will decrease as a function of age. This metabolic fate-mapping approach will enable the study of aging-related regeneration of cardiomyocytes. Furthermore, this new approach can be used not only for the myocardium but in other types of tissues such as skeletal muscle, brain and adipocytes. Finally, because this approach can be applied with stable, non-radioactive isotopes, long-term clinical studies of regenerative activity in human subjects may be enabled by this method. PUBLIC HEALTH RELEVANCE: Heart Failure, a leading cause of death and disability in the United States, is often due to the loss of heart muscle cells, with inadequate replacement by new heart cells. Understanding why heart cells don't regenerate in older age is a critical goal of cardiovascular science. This project will develop a new approach to measuring birth of new heart cells, allowing us to understand the effect of aging on the heart.